1. Field of the Invention
The present invention relates generally to the fields of molecular biology and protein chemistry of promoters. More specifically, the present invention relates to optimized core promoters, the design thereof and uses therefor.
2. Description of the Related Art
The regulation of gene expression at the level of transcription is a key control point for many cellular processes. In eukaryotes, there are tens of thousands of protein-coding genes, each of which has its own unique program of transcription. To understand how these transcriptional programs are encoded in the DNA template, it is necessary to understand the fundamental molecular mechanisms by which transcription is regulated.
Transcription by RNA polymerase II involves a network of factors that include sequence-specific DNA-binding proteins, transcriptional coregulators, chromatin remodeling factors, enzymes that covalently modify histones (and other proteins), and the basal transcriptional machinery. A proportion of the regulatory information that specifies the transcriptional program of each gene is encoded in promoters and enhancers. However, the ultimate target sequence of the vast array of factors that control the initiation of transcription is the core promoter (1-5).
The core promoter encompasses the RNA start site and directs the accurate initiation of transcription. Core promoters are typically about 50 nt in length, and consist of functional subregions termed core promoter elements. These core promoter elements are not universally present in all core promoters. Rather, core promoter elements confer the specific properties of each core promoter, such as the interactions of core promoters with enhancer elements. Some of these core promoter motifs are as follows.
The TATA box (6) is an A/T-rich sequence that is located approximately 26 to 31 nt upstream of the transcription start site. The TATA box is a recognition site for the binding of the TATA box-binding protein (TBP) subunit of TFIID. The TFIIB recognition element (BRE) is located immediately upstream of about 12% of TATA boxes (7). The initiator element (Inr) encompasses the transcription start site (8). The Inr is recognized by the TAF1 (TAFII250) and TAF2 (TAFII150) subunits of the TFIID complex (see, e.g., 9-12) as well as several other factors (discussed in 5).
The DPE is a downstream core promoter element that is located from +28 to +33 relative to the A+1 in the Inr motif (13-15). A typical DPE-dependent promoter has Inr and DPE motifs and lacks a TATA box. The spacing between the Inr and DPE motifs has been observed to be identical in all (˜30) DPE-dependent promoters examined thus far. The DPE is conserved from Drosophila to humans, and appears to be as common as the TATA box in Drosophila core promoters. TFIID binds cooperatively to the Inr and DPE motifs (13). Photocrosslinking analyses indicated that the TAF6 (TAF60) and TAF9 (TAF40) subunits of Drosophila TFIID are in close proximity to the DPE (14). Gel shift analyses further revealed sequence-specific binding of TAF6-TAF9 complexes to the DPE motif (16). In addition, TAF9 in vivo associates preferentially with the DPE-containing human IRF-1 promoter relative to a DPE-mutant version of the promoter (17).
There are significant differences in the mechanisms of transcription from DPE-versus TATA-dependent core promoters. For instance, NC2 (negative cofactor 2; also known as Dr1-Drap1), which had been initially identified as a repressor of transcription from TATA-containing promoters, was an activator of basal transcription from DPE-dependent core promoters (18). In addition, some enhancers activate transcription from DPE-dependent core promoters but not from TATA-dependent core promoters, and vice versa (19). Thus, the core promoter is a regulatory element that is an important component of enhancer function. The differences between DPE- versus TATA-directed transcription are further revealed by the analysis of the factors that are required for basal transcription. With TATA-dependent promoters, accurate initiation of transcription is mediated by RNA polymerase II along with the basal (‘general’) factors—TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. In contrast, transcription of DPE-dependent promoters does not occur with the same highly purified basal transcription factors in the presence or absence of purified recombinant dNC2.
Most scientists have attempted to enhance gene expression by optimization and/or modification of the elements that are bound by sequence-specific DNA-binding proteins that interact with proximal promoter and enhancer elements, which is useful and successful. However, the core promoter has been overlooked in such endeavors, largely because of a general lack of knowledge and appreciation of the core promoter. The synthesis of recombinant proteins has been widely employed in the biotech industry for the production of important pharmaceuticals such as insulin, human growth hormone, erythropoietin (EPO), and tissue plasminogen activator (tPA). Some proteins, such as insulin, are produced in bacteria while other proteins, e.g., EPO and tPA, are produced in metazoan cells (that is, multicellular animal cells, which include mammalian and insect cells) to achieve the posttranslational processing that is necessary for biological activity. The present invention is relevant to the latter category of recombinant proteins that are produced in metazoan cells.
There is a need in the art for improved production of recombinant proteins by focusing on the core promoter element, instead of upstream promoter and enhancer elements. Specifically, the prior art is deficient in optimized core promoters useful in methods of increasing protein production in metazoan cells. The present invention fulfills this long-standing need and desire in the art.